Targeted capture and sequencing identifies causative alleles in simplex and multiplex consanguineous Palestinian families with orofacial clefts. H. Shahin1, U. Sharaha1, M. K. Lee2, A. Watts2, M.-C. King2, J. van Aalst3, T. Walsh2 1) Dept Life Sci, Bethlehem Univ, Bethlehem, Palestinian Territory; 2) Division of Medical Genetics, University of Washington, Seattle, WA; 3) Division of Plastic and Reconstructive Surgery, University of North Carolina, Chapel Hill, NC.

   Orofacial clefts are among the most common of all human congenital birth defects. They are divided into two categories on the basis of embryological findings: cleft lip with or without cleft palate (CL/P) and cleft palate only (CP). CL/P affects between 1/2500 and 1/500 live births with considerable geographic variability in rates. Overall, approximately 70% of CL/P and 50% of CP are non-syndromic. Genes responsible for orofacial clefts were first identified by linkage and positional cloning studies of families and more recently by exome sequencing. In order to more efficiently identify causative alleles in genes known to cause orofacial clefts, we developed a targeted capture panel to sequence the coding exons and flanking splice sites of 137 genes. The capture size of 494kb enabled deep multiplexed sequencing while maintaining high coverage and detection of single base substitutions, indels and exonic copy number variants. Using this capture panel, we evaluated 44 Palestinian patients with orofacial clefts undergoing corrective surgeries, but with no prior genetic testing. Of the 44 patients, 20 were simplex cases with no family history of clefting, and 24 were probands of multiplex families. In four of the 20 simplex cases, we identified heterozygous mutations, either truncations or functionally validated damaging missense mutations, in GLI2, MSX1, SATB2, and TP63. In four of the 24 probands from multiplex families, we identified a homozygous missense mutation in COL2A1 (Gly855Ser). Genotyping all 41 members of these four extended kindreds revealed perfect co-segregation of COL2A1 p.G855S with clefting. Among homozygotes for COL2A1 p.G855S, there was considerable variability of clinical presentation in addition to clefting, including partial hearing loss, ocular problems, joints abnormalities, and dwarfism. Our approach enables comprehensive testing of all known clefting genes in a single assay and can be readily applied to large cohorts of patients with or without additional syndromic features. Sequencing in this way is likely to increase our knowledge of the type of clefting and additional clinical features associated with mutations in each gene.

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